Regulatory T cells are a CD4+ T cell subset with immunosuppressive activity that have been identified in humans and mice. These cells exert tolerance through a dominant mechanism that has obvious therapeutic implications for intervention in autoinflammatory diseases and transplantation. We have identified IL-10 producing CD4+ T cells in the normal mouse intestine that are reactive to enteric bacterial antigens and have Treg function in vitro and in vivo. We speculate that this population develops from mature, naive CD4 T cell precursors that recognize enteric bacterial antigens, and that IL-10 can be a useful marker with which to identify and study these cells. In this proposal, we will make use of a novel antigen-specific model of colitis based on the DO11.10 TCR transgenic mouse to examine the origin, function and maintenance of intestinal Tregs. We will also employ a new IL-10 reporter knock-in mouse to provide a functional marker that can be used to identify, isolate and characterize IL-10 producing Tregs in the intestinal tissues. These results will form the basis for comparative studies with Treg cells isolated from human intestinal tissues. The specific aims are to test four distinct, but related hypotheses: (1) IL-10 producing CD4 T cells (CD4+IL-10+) reactive to commensal bacterial antigens are a naturally occurring Treg population that exist in the intestinal mucosae and are the principal population responsible for intestinal immune homeostasis; (2) intestinal CD4+IL-10+ Tregs require the enteric flora for development and maintenance; (3) intestinal Tregs suppress the development and maintenance of colitogenic Th1 cells through bystander inhibition; and (4) human and mouse intestinal Tregs share common phenotypic and functional properties. These studies will advance our understanding of intestinal Tregs and will provide insights into how the immune system maintains tolerance to the enormous antigenic challenge represented by the enteric bacterial flora. Defective immunoregulation to the bacterial flora is a common feature of chronic intestinal inflammation in most murine models and is postulated to occur in patients with inflammatory bowel disease (IBD), such as Crohn's disease and ulcerativecolitis. These studies will provide a basis for manipulation of Treg function as a therapeutic approach to restoring dysregulated T cell responses in IBD.